REGULATION OF CELLULAR CA2+ BY YEAST VACUOLES

The role of vacuolar Ca2+ transport systems in regulating cellular Ca2 + was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate . The magnitude of the Ca2+ gradient generated by the vacuolar H+ grad ient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/ H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentr ation of 125 nM would give a vacuolar free Ca2+ concentration of simil ar to 30 mu M. The total vacuolar Ca2+ concentration is similar to 2 m M due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate from the vacuole is less than the growth rate indicating that the stea dy-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by grow th. Based on the kinetic parameters of vacuolar Ca2+ accumulation in v itro, the maximum rate of Ca2+ accumulation in vivo is expected to be similar to 0.2 nmol of Ca2+ min(-1) mg protein(-1), a rate that is sim ilar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concen tration increases from 0.1 mu M to 1-2 mu M as the extracellular Ca2concentration is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca 2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg b y increasing the rate of vacuolar Ca2+ accumulation but does not signi ficantly alter the cellular growth rate.